DIRECTED MOLECULAR EVOLUTION OF PROTEINS
1 INTRODUCTION 1 2 EVOLUTIONARY BIOTECHNOLOGY FROM IDEAS AND CONCEPTS TO EXPERIMENTS AND COMPUTER SIMULATIONS 5 21 EVOLUTION IN VIVO FROM NATURAL SELECTION TO POPULATION GENETICS 5 22 EVOLUTION IN VITRO FROM KINETIC EQUATIONS TO MAGIC MOLECULES 8 23 EVOLUTION IN SILICO FROM NEUTRAL NETWORKS TO MULTI-STABLE MOLECULES 16 24 SEQUENCE STRUCTURE MAPPINGS OF PROTEINS 25 25 CONCLUDING REMARKS 26 3 USING EVOLUTIONARY STRATEGIES TO INVESTIGATE THE STRUCTURE AND FUNCTION OF CHORISMATE MUTASES 29 31 INTRODUCTION 29 32 SELECTION VERSUS SCREENING 30 321 CLASSICAL SOLUTIONS TO THE SORTING PROBLEM 31 322 ADVANTAGES AND LIMITATIONS OF SELECTION 32 33 GENETIC SELECTION OF NOVEL CHORISMATE MUTASES 33 331 THE SELECTION SYSTEM 35 332 MECHANISTIC STUDIES 37 3321 ACTIVE SITE RESIDUES 37 3322 RANDOM PROTEIN TRUNCATION 42 333 STRUCTURAL STUDIES 44 3331 CONSTRAINTS ON INTERHELICAL LOOPS 44 ALTERING PROTEIN TOPOLOGY 46 334 3341 NEW QUATERNARY STRUCTURES 47 3342 STABLE MONOMERIC MUTASES 49 335 AUGMENTING WEAK ENZYME ACTIVITY 51 336 PROTEIN DESIGN 53 34 SUMMARY AND GENERAL PERSPECTIVES 57 4 CONSTRUCTION OF ENVIRONMENTAL LIBRARIES FOR FUNCTIONAL SCREENING OF ENZYME ACTIVITY 63 41 SAMPLE COLLECTION AND DNA ISOLATION FROM ENVIRONMENTAL SAMPLES 65 42 CONSTRUCTION OF ENVIRONMENTAL LIBRARIES 68 43 SCREENING OF ENVIRONMENTAL LIBRARIES 71 44 CONCLUSIONS 76 5 INVESTIGATION OF PHAGE DISPLAY FOR THE DIRECTED EVOLUTION OF ENZYMES 79 51 INTRODUCTION 79 52 THE PHAGE DISPLAY 79 53 PHAGE DISPLAY OF ENZYMES 81 531 THE EXPRESSION VECTORS 81 5311 FILAMENTOUS BACTERIOPHAGES 81 5312 OTHER PHAGES 83 532 PHAGE-ENZYMES 84 54 CREATING LIBRARIES OF MUTANTS 87 55 SELECTION OF PHAGE-ENZYMES 89 551 SELECTION FOR BINDING 89 552 SELECTION FOR CATALYTIC ACTIVITY 90 5521 SELECTION WITH SUBSTRATE OR PRODUCT ANALOGUES 90 5522 SELECTION WITH TRANSITION-STATE ANALOGUES 92 5523 SELECTION OF REACTIVE ACTIVE SITE RESIDUES BY AFFINITY LABELING 96 5524 SELECTION WITH SUICIDE SUBSTRATES 98 5525 SELECTIONS BASED DIRECTLY ON SUBSTRATE TRANSFORMATIONS 102 56 CONCLUSIONS 108 6 DIRECTED EVOLUTION OF BINDING PROTEINS BY CELL SURFACE DISPLAY: ANALYSIS OF THE SCREENING PROCESS 111 INTRODUCTION 111 61 62 LIBRARY CONSTRUCTION 113 621 MUTAGENESIS 113 622 EXPRESSION 114 63 MUTANT ISOLATION 115 631 DIFFERENTIAL LABELING 115 632 SCREENING 119 64 SUMMARY 124 ACKNOWLEDGMENTS 124 7 YEAST N-HYBRID SYSTEMS FOR MOLECULAR EVOLUTION 127 71 INTRODUCTION 127 72 TECHNICAL CONSIDERATIONS 130 721 YEAST TWO-HYBRID ASSAY 130 722 ALTERNATIVE ASSAYS 141 73 APPLICATIONS 147 731 PROTEIN-PROTEIN INTERACTIONS 147 732 PROTEIN-DNA INTERACTIONS 149 733 PROTEIN-RNA INTERACTIONS 150 734 PROTEIN-SMALL MOLECULE INTERACTIONS 153 74 CONCLUSION 155 8 ADVANCED SCREENING STRATEGIES FOR BIOCATALYST DISCOVERY 159 81 INTRODUCTION 159 82 SEMI-QUANTITATIVE SCREENING IN AGAR-PLATE FORMATS 161 83 SOLUTION-BASED SCREENING IN MICROPLATE FORMATS 164 84 ROBOTICS AND AUTOMATION 169 9 ENGINEERING PROTEIN EVOLUTION 177 91 INTRODUCTION 177 92 MECHANISMS OF PROTEIN EVOLUTION IN NATURE 178 921 GENE DUPLICATION 179 922 TANDEM DUPLICATION 180 BA-BARRELS 181 923 CIRCULAR PERMUTATION 182 924 OLIGOMERIZATION 183 925 GENE FUSION 184 926 DOMAIN RECRUITMENT 184 927 EXON SHUFFLING 186 93 ENGINEERING GENES AND GENE FRAGMENTS 187 931 PROTEIN FRAGMENTATION 188 932 RATIONAL SWAPPING OF SECONDARY STRUCTURE ELEMENTS AND DOMAINS 189 933 COMBINATORIAL GENE FRAGMENT SHUFFLING 190 934 MODULAR RECOMBINATION AND PROTEIN FOLDING 194 935 RATIONAL DOMAIN ASSEMBLY ENGINEERING ZINC FINGERS 199 936 COMBINATORIAL DOMAIN RECOMBINATION EXON SHUFFLING 200 94 GENE FUSION - FROM BI-TO MULTIFUNCTIONAL ENZYMES 203 941 END-TO-END GENE FUSIONS 203 942 GENE INSERTIONS 203 943 MODULAR DESIGN IN MULTIFUNCTIONAL ENZYMES 204 95 PERSPECTIVES 208 10 EXPLORING THE DIVERSITY OF HEME ENZYMES THROUGH DIRECTED EVOLUTION 215 101 INTRODUCTION 215 102 HEME PROTEINS 216 103 CYTOCHROMES P450 218 1031 INTRODUCTION 218 1031 MECHANISM 220 10321 THE CATALYTIC CYCLE 220 10322 UNCOUPLING 222 10323 PEROXIDE SHUNT PATHWAY 222 104 PEROXIDASES 223 1041 INTRODUCTION 223 1042 MECHANISM 223